Silencing NKD2 by Promoter Region Hypermethylation Promotes Esophageal Cancer Progression by Activating Wnt Signaling  Baoping Cao, MD, PhD, Weili Yang, MD, Yongshuai Jin, MD, Meiying Zhang, MS, Tao He, MD, Qimin Zhan, MD, James G. Herman, MD, Guanglin Zhong, MD, Mingzhou Guo, MD, PhD  Journal of Thoracic Oncology  Volume 11, Issue 11, Pages 1912-1926 (November 2016) DOI: 10.1016/j.jtho.2016.06.015 Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 The expression and methylation status of naked cuticle homolog 2 (NKD2) in esophageal cancer cells and normal esophageal mucosa (NE). (A) Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) shows NKD2 expression levels in esophageal cancer cell lines. KYSE450, KYSE30, KYSE150, KESE70, TE8, KYSE410, TE1, KYSE140, and COLO680 are esophageal cancer cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the internal control of RT-PCR. H2O is double-distilled water. Minus sign indicates absence of 5-aza-2′-deoxycytidine (5-AZA) and plus sign indicates presence of 5-AZA. (B) Methylation-specific polymerase chain reaction (MSP) results of NKD2 in esophageal cancer cell lines. U refers to unmethylated alleles, and M refers to methylated alleles. In vitro methylated DNA (IVD) serves as a methylation control; normal peripheral lymphocyte DNA (NL) serves as an unmethylation control. H2O is double-distilled water. (C) Bisulfite sequencing (BSSQ) results of NKD2. KYSE150, KYSE410, TE1, and KYSE450 are esophageal cancer cells. Double-headed arrow indicaets that the MSP PCR product spanned 103 base pairs (bp) in NKD2. Bisulfite sequencing focused on a 287-bp region of the CpG island (–287 bp to +38 bp) across the NKD2 transcription start site (TSS). Filled circles are methylated CpG sites, and open circles are unmethylated CpG sites. Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Methylation status and expression of naked cuticle homolog 2 (NKD2) in primary esophageal cancer samples. (A) Methylation-specific polymerase chain reaction (MSP) results of NKD2 in normal esophageal mucosa (NE). (B) Representative results of MSP for NKD2 in primary esophageal cancer samples (EC). (C) Representative immunohistochemical analysis results showing NKD2 expression in esophageal cancer and matched adjacent tissue samples (upper images: ×100; lower images: ×400). (D) NKD2 expression scores are shown as box plots, with horizontal lines representing the median score; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively, and vertical bars represent the range of data. The expression level of NKD2 was significantly different between adjacent tissue and esophageal cancer samples (***p < 0.001). (E) The bar diagram shows the expression and DNA methylation status of NKD2 in different cancer samples. Reduced expression of NKD2 was significantly associated with promoter region methylation (**p < 0.01). Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Naked cuticle homolog 2 (NKD2) inhibits esophageal cancer cell proliferation. (A) Growth curves represent the cell viability analyzed by the methyl thiazolyl tetrazolium assay in KYSE150 and TE1 cells in which NKD2 was reexpressed and unexpressed. The experiments were performed in triplicate (*p < 0.05 and ***p < 0.001). (B) Colony formation results show that colony number was reduced by reexpression of NKD2 in KYSE150 and TE1 cells. Each experiment was repeated three times. The average number of tumor clones is represented by a bar diagram (***p < 0.001). (C) Cell phase distribution in KYSE150 and TE1 cells in which NKD2 was unexpressed and reexpressed. The ratio is presented by a bar diagram. Each experiment was repeated three times (**p < 0.01 and ***p < 0.001). G0/G1, G0/G1 phase; S, S phase; G2/M, G2/M phase. Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Restoration of naked cuticle homolog 2 (NKD2) expression inhibits cell migration and invasion. (A) Cell migration in KYSE150 and TE1 cells in which NKD2 was unexpressed and reexpressed. The ratio is presented by a bar diagram. Each experiment was repeated three times (***p < 0.001). (B) Cell invasion in KYSE150 and TE1 cells in which NKD2 was unexpressed and reexpressed. The ratio is presented by a bar diagram. Each experiment was repeated three times (***p < 0.001). (C) The expression levels of NKD2, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-7 (MMP-7), and matrix metalloproteinase-9 (MMP-9) were detected by Western blot in KYSE150 and TE1 cells in which NKD2 was unexpressed and reexpressed. Knockdown of NKD2 by small interfering RNA (siRNA) was performed to validate the results in KYSE450 cells in which NKD2 was highly expressed. (D) Representative images of immunohistochemical analysis for NKD2, MMP2, and MMP9 in human esophageal squamous cell cancer. The expression levels of NKD2, MMP2, and MMP9 were evaluated by the German semiquantitative scoring system. The correlation of NKD2 and MMP2, or NKD2 and MMP9, was analyzed by Pearson correlation coefficient. The x axis represents levels of NKD2 and the y axis represents levels of MMP2 or MMP9. NC, KYSE450 cells in which NKD2 is highly expressed; SiNKD2, KYSE450 cells in which NKD2 is knocked down by siRNA. Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 5 Naked cuticle homolog 2 (NKD2) inhibits canonical Wnt signaling in human esophageal cancer cells. (A) The expression levels of β-catenin, cyclin D1, and c-myc were reduced, and the level of phosphorylated β-catenin (p-β-catenin) increased after reexpression of NKD2 in KYSE150 and TE1 cells. (B) The level of p-β-catenin was reduced and the expression levels of β-catenin, c-myc, and cyclin D1 increased after knockdown of NKD2 by small interfering RNA (siRNA) in KYSE450 cells. (C) Representative images of immunohistochemical analysis for NKD2 and phosphorylated β-catenin (p-β-catenin) in human esophageal squamous cell cancer. The expression levels of NKD2 and p-β-catenin were evaluated by the German semiquantitative scoring system. The correlation of NKD2 and p-β-catenin was analyzed with Pearson's correlation coefficient. The x axis represents levels of NKD2, and the y axis represents levels of p-β-catenin. NC, KYSE450 cells in which NKD2 is highly expressed; SiNKD2, KYSE450 cells in which NKD2 has been knocked down by siRNA. Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 6 Naked cuticle homolog 2 (NKD2) suppresses esophageal cancer cell growth in xenograft mice. (A) Representative burdened nude mice with NKD2 reexpressed and unexpressed in KYSE150 cells and NKD2 expressed and knocked down in KYSE450 cells. The tumor locations are shown by red arrowhead. (B) Subcutaneous tumor growth curves for xenograft mice burdened with KYSE150 cells in which NKD2 is unexpressed and reexpressed and KYSE450 cells is which NKD2 is expressed and knocked down at different times (***p < 0.001). (C) Tumor weight in nude mice at the 24th day after inoculation with KYSE150 cells in which NKD2 is unexpressed and reexpressed and KYSE450 cells in which NKD2 is expressed and knocked down. Bars indicate mean of six mice (***p < 0.001). (D) Representative photographs of immunohistochemical analysis for NKD2, β-catenin, and p-β-catenin in xenografts. Staining of NKD2 and p-β-catenin was found in KYSE150 cell xenografts in which NKD2 is reexpressed. Staining of total β-catenin was reduced in KYSE150 cell xenografts in which NKD2 is reexpressed. Magnification: ×400. NC, KYSE450 cells in which NKD2 is highly expressed; SiNKD2, KYSE450 cells in which NKD2 has been knocked down by small interfering RNA. Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 1 Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 2 Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 3 Journal of Thoracic Oncology 2016 11, 1912-1926DOI: (10.1016/j.jtho.2016.06.015) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions